HPV-YAP1 oncogenic alliance drives malignant transformation of fallopian tube epithelial cells.

High grade serous ovarian carcinoma (HGSOC) is the most common and aggressive ovarian malignancy. Accumulating evidence indicates that HGSOC may originate from human fallopian tube epithelial cells (FTECs), although the exact pathogen(s) and/or molecular mechanism underlying the malignant transformation of FTECs is unclear. Here we show that human papillomavirus (HPV), which could reach FTECs via retrograde menstruation or sperm-carrying, interacts with the yes-associated protein 1 (YAP1) to drive the malignant transformation of FTECs. HPV prevents FTECs from natural replicative and YAP1-induced senescence, thereby promoting YAP1-induced malignant transformation of FTECs. HPV also stimulates proliferation and drives metastasis of YAP1-transformed FTECs. YAP1, in turn, stimulates the expression of the putative HPV receptors and suppresses the innate immune system to facilitate HPV acquisition. These findings provide critical clues for developing new strategies to prevent and treat HGSOC.

Adolescent exposure to a mixture of per- and polyfluoroalkyl substances (PFAS) depletes the ovarian reserve, increases ovarian fibrosis, and alters the hippo pathway in adult female mice.

Per- and polyfluoroalkyl substances (PFAS) are synthetic chemicals known for their environmental persistence and resistance to biodegradation. This study investigated the impact of adolescent exposure to a PFAS mixture on adult ovarian function. Female CD-1 mice were orally exposed to vehicle control or a PFAS mixture (comprised of perfluorooctanoic acid [PFOA], perfluorooctanesulfonic acid [PFOS], undecafluoro-2-methyl-3-oxahexanoic acid [GenX/HFPO-DA], and perfluorobutanesulfonic acid [PFBS]) for 15 days. After a 42-day recovery period, reproductive hormones, ovarian fibrosis, and ovarian gene and protein expression were analyzed using ELISA, Picrosirius red (PSR) staining, qPCR, and immunoblotting, respectively. Results revealed that PFAS exposure did not affect adult body or organ weight, although ovarian weight slightly decreased. PFAS exposed mice exhibited a disturbed estrous cycle, with less time spent in proestrus than control mice. Follicle counting indicated a reduction in primordial and primary follicles. Serum analysis revealed no changes in steroid hormones, follicle-stimulating hormone, or anti-Müllerian hormone, but a significant increase in luteinizing hormone was observed in PFAS-treated mice. Ovaries collected from PFAS treated mice had increased mRNA transcripts for steroidogenic enzymes and fatty acid synthesis-related genes. PFAS exposure also increased collagen content in the ovary. Additionally, serum tumor necrosis factor-α levels were higher in PFAS treated mice. Finally, transcripts and protein abundance for Hippo pathway components were upregulated in the ovaries of the PFAS treated mice. Overall, these findings suggest that adolescent exposure to PFAS can disrupt ovarian function in adulthood.

Characterization of m6A Modifiers and RNA Modifications in Uterine Fibroids.

Uterine leiomyoma or fibroids are prevalent noncancerous tumors of the uterine muscle layer, yet their origin and development remain poorly understood. We analyzed RNA expression profiles of 15 epigenetic mediators in uterine fibroids compared to myometrium using publicly available RNA sequencing (RNA-seq) data. To validate our findings, we performed RT-qPCR on a separate cohort of uterine fibroids targeting these modifiers confirming our RNA-seq data. We then examined protein profiles of key N6-methyladenosine (m6A) modifiers in fibroids and their matched myometrium, showing no significant differences in concordance with our RNA expression profiles. To determine RNA modification abundance, mRNA and small RNA from fibroids and matched myometrium were analyzed by ultra-high performance liquid chromatography-mass spectrometry identifying prevalent m6A and 11 other known modifiers. However, no aberrant expression in fibroids was detected. We then mined a previously published dataset and identified differential expression of m6A modifiers that were specific to fibroid genetic subtype. Our analysis also identified m6A consensus motifs on genes previously identified to be dysregulated in uterine fibroids. Overall, using state-of-the-art mass spectrometry, RNA expression, and protein profiles, we characterized and identified differentially expressed m6A modifiers in relation to driver mutations. Despite the use of several different approaches, we identified limited differential expression of RNA modifiers and associated modifications in uterine fibroids. However, considering the highly heterogenous genomic and cellular nature of fibroids, and the possible contribution of single molecule m6A modifications to fibroid pathology, there is a need for greater in-depth characterization of m6A marks and modifiers in a larger and diverse patient cohort.

Interferon-tau infusion into the ovine corpus luteum delays luteolysis†.

Conceptus-derived interferon-tau (IFNT) initiates maternal recognition of pregnancy in ewes by paracrine actions on the endometrium and endocrine action on the corpus luteum (CL). To examine the effect of IFNT on the CL without inducing IFN-stimulated genes (ISGs) in the endometrium, recombinant ovine IFNT (roIFNT) or bovine serum albumin was delivered directly into CLs via osmotic pumps at a rate of 10, 50, or 100 ng/h from days 9 to 12 of the estrous cycle. Endometrial and CL samples were collected on day 12. 50 ng/h of roIFNT induced ISG15 in the CL on day 12 without affecting endometrial ISG15 concentrations. In a second experiment, roIFNT (50 ng/h) was infused into the CL from days 10 to 17 of the estrous cycle and serum samples were collected daily. Serum progesterone concentrations were significantly higher from days 15 to 17 in roIFNT-infused ewes compared to controls. Levels of LHCGR, STAR, CYP11A1, HSL, OPA1, and protein kinase A mRNA and proteins were higher in the roIFNT-infused CLs compared to the controls. Levels of ISG15 and MX1 mRNA increased in the CLs of roIFNT-infused ewes but not in the endometrium. Endometrial ESR1 mRNA and protein concentrations were higher in the controls compared to roIFNT-infused ewes. In conclusion, intra-luteal delivery of roIFNT induced ISGs, stabilized steroidogenesis in the CL, and delayed luteolysis without inducing endometrial IFN-stimulated genes. Inhibition of ESR1 in the endometrium of roIFNT-infused ewes was observed suggesting that direct delivery of IFNT to the CL has an additional anti-luteolytic effect on the endometrium.

Unraveling the role of lipid droplets and perilipin 2 in bovine luteal cells.

Steroidogenic tissues contain cytosolic lipid droplets that are important for steroidogenesis. Perilipin 2 (PLIN2), a structural coat protein located on the surface of lipid droplets in mammalian cells, plays a crucial role in regulating lipid droplet formation and contributing to various cellular processes such as lipid storage and energy homeostasis. Herein, we examine the role that PLIN2 plays in regulating progesterone synthesis in the bovine corpus luteum. Utilizing gene array databases and Western blotting, we have delineated the expression pattern of PLIN2 throughout the follicular to luteal transition. Our findings reveal the presence of PLIN2 in both ovarian follicular and steroidogenic luteal cells, demonstrating an increase in its levels as follicular cells transition into the luteal phase. Moreover, the depletion of PLIN2 via siRNA enhanced progesterone production in small luteal cells, whereas adenovirus-mediated overexpression of both PLIN2 and Perilipin 3 (PLIN3) induced an increase in cytosolic lipid droplet accumulation and decreased hormone-induced progesterone synthesis in these cells. Lastly, in vivo administration of the luteolytic hormone prostaglandin F2α resulted in an upregulation of PLIN2 mRNA and protein expression, accompanied by a decline in serum progesterone. Our findings highlight the pivotal role of PLIN2 in regulating progesterone synthesis in the bovine corpus luteum, as supported by its dynamic expression pattern during the follicular to luteal transition and its responsiveness to luteotropic and luteolytic hormones. We suggest PLIN2 as a potential therapeutic target for modulating luteal function.

An environmentally relevant mixture of per- and polyfluoroalkyl substances (PFAS) impacts proliferation, steroid hormone synthesis, and gene transcription in primary human granulosa cells.

Per- and polyfluoroalkyl substances (PFAS) are a group of synthetic chemicals that are resistant to biodegradation and are environmentally persistent. PFAS are found in many consumer products and are a major source of water and soil contamination. This study investigated the effects of an environmentally relevant PFAS mixture (perfluorooctanoic acid [PFOA], perfluorooctanesulfonic acid [PFOS], perfluorohexanesulfonic acid [PFHxS]) on the transcriptome and function of human granulosa cells (hGCs). Primary hGCs were harvested from follicular aspirates of healthy, reproductive-age women who were undergoing oocyte retrieval for in vitro fertilization. Liquid Chromatography with tandem mass spectrometry (LC/MS-MS) was performed to identify PFAS compounds in pure follicular fluid. Cells were cultured with vehicle control or a PFAS mixture (2 nM PFHxS, 7 nM PFOA, 10 nM PFOS) for 96 h. Analyses of cell proliferation/apoptosis, steroidogenesis, and gene expression were measured via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays/immunofluorescence, ELISA/western blotting, and RNA sequencing/bioinformatics, respectively. PFOA, PFOS, and PFHxS were detected in 100% of follicle fluid samples. Increased cell proliferation was observed in hGCs treated with the PFAS mixture with no impacts on cellular apoptosis. The PFAS mixture also altered steroid hormone synthesis, increasing both follicle-stimulating hormone-stimulated and basal progesterone secretion and concomitant upregulation of STAR protein. RNA sequencing revealed inherent differences in transcriptomic profiles in hGCs after PFAS exposure. This study demonstrates functional and transcriptomic changes in hGCs after exposure to a PFAS mixture, improving our knowledge about the impacts of PFAS exposures and female reproductive health. These findings suggest that PFAS compounds can disrupt normal granulosa cell function with possible long-term consequences on overall reproductive health.

Neddylation inhibition affects early embryonic development by disrupting maternal-to-zygotic transition and mitochondrial function in mice.

Post-translational modifications (PTMs) are critical for early development in mice because early cleavage-stage embryos are characterized by transcriptional inactivity. Neddylation is an important ubiquitin-like PTM that regulates multiple biophysical processes. However, the exact roles of neddylation in regulating early embryonic development remain largely unknown. In the present study, we found that inhibition of neddylation by specific inhibitor MLN4924 led to severe arrest of early embryonic development. Transcriptomic analysis showed that neddylation inhibition changed the expression of 3959 genes at the 2-cell stage. Importantly, neddylation inhibition blocked zygotic genome activation and maternal mRNA degradation, thus disrupting the maternal-to-zygotic transition. Moreover, inhibition of neddylation induced mitochondrial dysfunction including aberrant mitochondrial distribution, decreased mitochondrial membrane potential, and reduced ATP content. Further analysis showed that inhibition of neddylation resulted in the accumulation of reactive oxygen species and superoxide anion, thereby resulting in oxidative stress and severe DNA damage at the 2-cell stage. Overall, this study demonstrates that neddylation is vital for early embryonic development in mice. Our findings suggest that proper neddylation regulation is essential for the timely inter-stage transition during early embryonic development.

Central Role for Glycolysis and Fatty Acids in LH-responsive Progesterone Synthesis.

Progesterone production by the corpus luteum is fundamental for establishing and maintaining pregnancy. The pituitary gonadotropin luteinizing hormone (LH) is recognized as the primary stimulus for luteal formation and progesterone synthesis, regardless of species. Previous studies demonstrated an elevation in abundance of genes related to glucose and lipid metabolism during the follicular to luteal transition. However, the metabolic phenotype of these highly steroidogenic cells has not been studied. Herein, we determined acute metabolic changes induced by LH in primary luteal cells and defined pathways required for progesterone synthesis. Untargeted metabolomics analysis revealed that LH induces rapid changes in vital metabolic pathways, including glycolysis, tricarboxylic acid (TCA) cycle, pentose phosphate pathway, de novo lipogenesis, and hydrolysis of phospholipids. LH stimulated glucose uptake, enhanced glycolysis, and flux of [U- 13 C 6 ]-labeled glucose-derived carbons into metabolic branches associated with adenosine 5'-triphosphate (ATP) and NADH/NADPH production, synthesis of nucleotides, proteins, and lipids, glycosylation of proteins or lipids, and redox homeostasis. Selective use of small molecule inhibitors targeting the most significantly changed pathways, such as glycolysis, TCA cycle, and lipogenesis, uncovered cellular metabolic routes required for LH-stimulated steroidogenesis. Furthermore, LH via the protein kinase A (PKA) pathway triggered post- translational modification of acetyl-CoA carboxylase alpha (ACACA) and ATP citrate lyase (ACLY), enzymes involved in de novo synthesis of fatty acids. Inhibition of ACLY and fatty acid transport into mitochondria reduced LH-stimulated ATP, cAMP production, PKA activation, and progesterone synthesis. Taken together, these findings reveal novel hormone-sensitive metabolic pathways essential for maintaining LHCGR/PKA signaling and steroidogenesis in ovarian luteal cells. Significance: The establishment and maintenance of pregnancy require a well-developed corpus luteum, an endocrine gland within the ovary that produces progesterone. Although there is increased awareness of intracellular signaling events initiating the massive production of progesterone during the reproductive cycle and pregnancy, there are critical gaps in our knowledge of the metabolic and lipidomic pathways required for initiating and maintaining luteal progesterone synthesis. Here, we describe rapid, hormonally triggered metabolic pathways, and define metabolic targets crucial for progesterone synthesis by ovarian steroidogenic cells. Understanding hormonal control of metabolic pathways may help elucidate approaches for improving ovarian function and successful reproduction or identifying metabolic targets for developing nonhormonal contraceptives.

Perfluorooctanoic acid (PFOA) inhibits steroidogenesis and mitochondrial function in bovine granulosa cells in vitro.

Perfluorooctanoic acid (PFOA) is a persistent environmental contaminant. Due to the ubiquitous presence of PFOA in the environment, the impacts of PFOA exposure not only affect human reproductive health but may also affect livestock reproductive health. The focus of this study was to determine the effects of PFOA on the physiological functions of bovine granulosa cells in vitro. Primary bovine granulosa cells were exposed to 0, 4, and 40 µM PFOA for 48 and 96 h followed by analysis of granulosa cell function including cell viability, steroidogenesis, and mitochondrial activity. Results revealed that PFOA inhibited steroid hormone secretion and altered the expression of key enzymes required for steroidogenesis. Gene expression analysis revealed decreases in mRNA transcripts for CYP11A1, HSD3B, and CYP19A1 and an increase in STAR expression after PFOA exposure. Similarly, PFOA decreased levels of CYP11A1 and CYP19A1 protein. PFOA did not impact live cell number, alter the cell cycle, or induce apoptosis, although it reduced metabolic activity, indicative of mitochondrial dysfunction. We observed that PFOA treatment caused a loss of mitochondrial membrane potential and increases in PINK protein expression, suggestive of mitophagy and mitochondrial damage. Further analysis revealed that these changes were associated with increased levels of reactive oxygen species. Expression of autophagy related proteins phosphoULK1 and LAMP2 were increased after PFOA exposure, in addition to an increased abundance of lysosomes, characteristic of increased autophagy. Taken together, these findings suggest that PFOA can negatively impact granulosa cell steroidogenesis via mitochondrial dysfunction.

Identification of Lipids and Cytokines in Plasma and Follicular Fluid before and after Follicle-Stimulating Hormone Stimulation as Potential Markers for Follicular Maturation in Cattle.

The process of follicle maturation leading to ovulation is a key milestone in female fertility. It is known that circulating lipids and cytokines play a role in the follicle's ability to go through follicular maturation and the ovulatory processes. However, the specific mechanisms are not well understood. We posit that dysregulation of granulosa cells influences the ovarian environment, which tries to adapt by changing released lipids and cytokines to achieve follicular maturation. Eleven non-lactating adult females underwent estrus synchronization with two injections of PGF2α 14 days apart. Daily blood samples were collected for 28 days to monitor steroid hormone production after the second injection. To understand the potential impacts of lipids and cytokines during ovulation, a low-dose FSH stimulation (FSHLow) was performed after resynchronization of cows, and daily blood samples were collected for 14 days to monitor steroid hormone production until ovariectomies. The lipidomic analysis demonstrated increased circulating diacylglycerides and triacylglycerides during the mid-luteal phase and after FSHLow treatment. Cholesteryl esters decreased in circulation but increased in follicular fluid (FF) after FSHLow. Increased circulating concentrations of TNFα and reduced CXCL9 were observed in response to FSHLow. Therefore, specific circulating lipids and cytokines may serve as markers of normal follicle maturation.

Mechanisms of angioregression of the corpus luteum.

The corpus luteum is a transient ovarian endocrine gland that produces the progesterone necessary for the establishment and maintenance of pregnancy. The formation and function of this gland involves angiogenesis, establishing the tissue with a robust blood flow and vast microvasculature required to support production of progesterone. Every steroidogenic cell within the corpus luteum is in direct contact with a capillary, and disruption of angiogenesis impairs luteal development and function. At the end of a reproductive cycle, the corpus luteum ceases progesterone production and undergoes rapid structural regression into a nonfunctional corpus albicans in a process initiated and exacerbated by the luteolysin prostaglandin F2α (PGF2α). Structural regression is accompanied by complete regression of the luteal microvasculature in which endothelial cells die and are sloughed off into capillaries and lymphatic vessels. During luteal regression, changes in nitric oxide transiently increase blood flow, followed by a reduction in blood flow and progesterone secretion. Early luteal regression is marked by an increased production of cytokines and chemokines and influx of immune cells. Microvascular endothelial cells are sensitive to released factors during luteolysis, including thrombospondin, endothelin, and cytokines like tumor necrosis factor alpha (TNF) and transforming growth factor ß 1 (TGFB1). Although PGF2α is known to be a vasoconstrictor, endothelial cells do not express receptors for PGF2α, therefore it is believed that the angioregression occurring during luteolysis is mediated by factors downstream of PGF2α signaling. Yet, the exact mechanisms responsible for angioregression in the corpus luteum remain unknown. This review describes the current knowledge on angioregression of the corpus luteum and the roles of vasoactive factors released during luteolysis on luteal vasculature and endothelial cells of the microvasculature.

Luteal Lipid Droplets: A Novel Platform for Steroid Synthesis.

Progesterone is an essential steroid hormone that is required to initiate and maintain pregnancy in mammals and serves as a metabolic intermediate in the synthesis of endogenously produced steroids, including sex hormones and corticosteroids. Steroidogenic luteal cells of the corpus luteum have the tremendous capacity to synthesize progesterone. These specialized cells are highly enriched with lipid droplets that store lipid substrate, which can be used for the synthesis of steroids. We recently reported that hormone-stimulated progesterone synthesis by luteal cells requires protein kinase A-dependent mobilization of cholesterol substrate from lipid droplets to mitochondria. We hypothesize that luteal lipid droplets are enriched with steroidogenic enzymes and facilitate the synthesis of steroids in the corpus luteum. In the present study, we analyzed the lipid droplet proteome, conducted the first proteomic analysis of lipid droplets under acute cyclic adenosine monophosphate (cAMP)-stimulated conditions, and determined how specific lipid droplet proteins affect steroidogenesis. Steroidogenic enzymes, cytochrome P450 family 11 subfamily A member 1 and 3 beta-hydroxysteroid dehydrogenase (HSD3B), were highly abundant on lipid droplets of the bovine corpus luteum. High-resolution confocal microscopy confirmed the presence of active HSD3B on the surface of luteal lipid droplets. We report that luteal lipid droplets have the capacity to synthesize progesterone from pregnenolone. Lastly, we analyzed the lipid droplet proteome following acute stimulation with cAMP analog, 8-Br-cAMP, and report increased association of HSD3B with luteal lipid droplets following stimulation. These findings provide novel insights into the role of luteal lipid droplets in steroid synthesis.

Characterization of m6A modifiers and RNA modifications in uterine fibroids.

Uterine leiomyoma or fibroids are the most common prevalent noncancerous tumors of the uterine muscle layer. Common symptoms associated with fibroids include pelvic pain, heavy menstrual bleeding, anemia, and pelvic pressure. These tumors are a leading cause of gynecological care but lack long-term therapy as the origin and development of fibroids are not well understood. Several next-generation sequencing technologies have been performed to identify the underlying genetic and epigenetic basis of fibroids. However, there remains a systemic gap in our understanding of molecular and biological process that define uterine fibroids. Recent epitranscriptomics studies have unraveled RNA modifications that are associated with all forms of RNA and are thought to influence both normal physiological functions and the progression of diseases. We quantified RNA expression profiles by analyzing publicly available RNA-seq data for 15 known epigenetic mediators to identify their expression profile in uterine fibroids compared to myometrium. To validate our findings, we performed RT-qPCR on a separate cohort of uterine fibroids targeting these modifiers confirming our RNA-seq data. We then examined protein profiles of key m6A modifiers in fibroids and their matched myometrium. In concordance with our RNA expression profiles, no significant differences were observed in these proteins in uterine fibroids compared to myometrium. To determine abundance of RNA modifications, mRNA and small RNA from fibroids and matched myometrium were analyzed by UHPLC MS/MS. In addition to the prevalent N6-methyladenosine (m6A), we identified 11 other known modifiers but did not identify any aberrant expression in fibroids. We then mined a previously published dataset and identified differential expression of m6A modifiers that were specific to fibroid genetic sub-type. Our analysis also identified m6A consensus motifs on genes previously identified to be dysregulated in uterine fibroids. Overall, using state-of-the-art mass spectrometry, RNA expression and protein profiles, we characterized and identified differentially expressed m6A modifiers in relation to driver mutations. Despite the use of several different approaches, we identified limited differential expression of RNA modifiers and associated modifications in uterine fibroids. However, considering the highly heterogenous genomic and cellular nature of fibroids, and the possible contribution of single molecule m6A modifications to fibroid pathology, there is a need for greater in-depth characterization of m6A marks and modifiers in a larger and varied patient cohort.

Basic fibroblast growth factor induces proliferation and collagen production by fibroblasts derived from the bovine corpus luteum†.

Cyclic regression of the ovarian corpus luteum, the endocrine gland responsible for progesterone production, involves rapid matrix remodeling. Despite fibroblasts in other systems being known for producing and maintaining extracellular matrix, little is known about fibroblasts in the functional or regressing corpus luteum. Vast transcriptomic changes occur in the regressing corpus luteum, among which are reduced levels of vascular endothelial growth factor A (VEGFA) and increased expression of fibroblast growth factor 2 (FGF2) after 4 and 12 h of induced regression, when progesterone is declining and the microvasculature is destabilizing. We hypothesized that FGF2 activates luteal fibroblasts. Analysis of transcriptomic changes during induced luteal regression revealed elevations in markers of fibroblast activation and fibrosis, including fibroblast activation protein (FAP), serpin family E member 1 (SERPINE1), and secreted phosphoprotein 1 (SPP1). To test our hypothesis, we treated bovine luteal fibroblasts with FGF2 to measure downstream signaling, type 1 collagen production, and proliferation. We observed rapid and robust phosphorylation of various signaling pathways involved in proliferation, such as ERK, AKT, and STAT1. From our longer-term treatments, we determined that FGF2 has a concentration-dependent collagen-inducing effect, and that FGF2 acts as a mitogen for luteal fibroblasts. FGF2-induced proliferation was greatly blunted by inhibition of AKT or STAT1 signaling. Our results suggest that luteal fibroblasts are responsive to factors that are released by the regressing bovine corpus luteum, an insight into the contribution of fibroblasts to the microenvironment in the regressing corpus luteum.

Prostaglandin F2α regulates mitochondrial dynamics and mitophagy in the bovine corpus luteum.

Prostaglandins are arachidonic acid-derived lipid mediators involved in numerous physiological and pathological processes. PGF2α analogues are therapeutically used for regulating mammalian reproductive cycles and blood pressure, inducing term labor, and treating ocular disorders. PGF2α exerts effects via activation of calcium and PKC signaling, however, little is known about the cellular events imposed by PGF2α signaling. Here, we explored the early effects of PGF2α on mitochondrial dynamics and mitophagy in the bovine corpus luteum employing relevant and well characterized in vivo and in vitro approaches. We identified PKC/ERK and AMPK as critical protein kinases essential for activation of mitochondrial fission proteins, DRP1 and MFF. Furthermore, we report that PGF2α elicits increased intracellular reactive oxygen species and promotes receptor-mediated activation of PINK-Parkin mitophagy. These findings place the mitochondrium as a novel target in response to luteolytic mediator, PGF2α. Understanding intracellular processes occurring during early luteolysis may serve as a target for improving fertility.

FHL2 deficiency impairs follicular development and fertility by attenuating EGF/EGFR/YAP signaling in ovarian granulosa cells.

Female subfertility is an increasing reproductive issue worldwide, which is partially related to abnormal ovarian follicular development. Granulosa cells (GCs), by providing the necessary physical support and microenvironment for follicular development, play critical roles in maintaining female fertility. We previously showed that ectopic expression of four and a half LIM domains 2 (FHL2) promoted ovarian granulosa cell tumor progression. However, its function in follicular development and fertility remains unknown. Here, we confirmed that FHL2 is highly expressed in human and mouse ovaries. FHL2 immunosignals were predominantly expressed in ovarian GCs. A Fhl2 knockout (KO) mouse model was generated to examine its roles in follicular development and fertility. Compared with wildtype, knockout of Fhl2 significantly decreased female litter size and offspring number. Furthermore, Fhl2 deficiency reduced ovarian size and impaired follicular development. RNA-sequencing analysis of GCs isolated from either KO or WT mice revealed that, Fhl2 deletion impaired multiple biological functions and signaling pathways, such as Ovarian Putative Early Atresia Granulosa Cell, ErbB, Hippo/YAP, etc. In vitro studies confirmed that FHL2 silencing suppressed GCs growth and EGF-induced GCs proliferation, while its overexpression promoted GC proliferation and decreased apoptosis. Mechanistic studies indicated that FHL2, via forming complexes with transcriptional factors AP-1 or NF-κB, regulated Egf and Egfr expression, respectively. Besides, FHL2 depletion decreased YAP1 expression, especially the active form of YAP1 (nuclear YAP1) in GCs of growing follicles. EGF, serving as an autocrine/paracrine factor, not only induced FHL2 expression and nuclear accumulation, but also stimulated YAP1 expression and activation. Collectively, our study suggests that FHL2 interacts with EGFR and Hippo/YAP signaling to regulate follicular development and maintain fertility. This study illuminates a novel mechanism for follicular development and a potential therapeutic target to address subfertility.

Perfluorooctanoic acid (PFOA) promotes follicular growth and alters expression of genes that regulate the cell cycle and the Hippo pathway in cultured neonatal mouse ovaries.

Perfluorooctanoic acid (PFOA) is a synthetic chemical resistant to biodegradation and is environmentally persistent. PFOA is found in many consumer products and is a major source of water contamination. While PFOA has been identified as a contaminant of concern for reproductive health, little is known about the effects of PFOA on ovarian follicular development and growth. Recent evidence indicates that the Hippo pathway is an important regulator of ovarian physiology. Here, we investigated the effects of PFOA on ovarian folliculogenesis during the neonatal period of development and potential impacts on the Hippo signaling pathway. Post-natal day 4 (PND4) neonatal ovaries from CD-1 mice were cultured with control medium (DMSO <0.01% final concentration) or PFOA (50 µM or 100 µM). After 96 h, ovaries were collected for histological analysis of folliculogenesis, gene and protein expression, and immunostaining. Results revealed that PFOA (50 µM) increased the number of secondary follicles, which was accompanied by increases in mRNA transcripts and protein of marker of proliferation marker Ki67 with no impacts on apoptosis markers Bax, Bcl2, or cleaved caspase-3. PFOA treatment (50 µM and 100 µM) stimulated an upregulation of transcripts for cell cycle regulators Ccna2, Ccnb2, Ccne1, Ccnd1, Ccnd2, and Ccnd3. PFOA also increased abundance of transcripts of Hippo pathway components Mst1/2, Lats1, Mob1b, Yap1, and Taz, as well as downstream Hippo pathway targets Areg, Amotl2, and Cyr61, although it decreased transcripts for anti-apoptotic Birc5. Inhibition of the Hippo pathway effector YAP1 with Verteporfin resulted in the attenuation of PFOA-induced follicular growth and proliferation. Together, these findings suggest that occupationally relevant levels of PFOA (50 µM) can stimulate follicular activation in neonatal ovaries potentially through activation of the Hippo pathway.

Human papillomavirus targets the YAP1-LATS2 feedback loop to drive cervical cancer development.

Human papillomavirus (HPV) infection is very common in sexually active women, but cervical cancer only develops in a small fraction of HPV-infected women, suggesting that unknown intrinsic factors associated with the unique genetic/genomic background of the high-risk population play a critical role in cervical carcinogenesis. Although our previous studies have identified the hyperactivated YAP1 oncogene as a critical contributor to cervical cancer, the molecular mechanism by which YAP1 drives cervical cancer is unknown. In the present study, we found that although the hyperactivated YAP1 caused a malignant transformation of immortalized cervical epithelial cells, it induced cellular senescence in cultures of primary human cervical epithelial cells (HCvECs). However, the hyperactivated YAP1 induced malignant transformation of HCvECs in the presence of high-risk HPV E6/E7 proteins, suggesting that the hyperactivated YAP1 synergizes with HPV to initiate cervical cancer development. Our mechanistic studies demonstrate that YAP1, via up-regulating LATS2, formed a YAP1-LATS2 negative feedback loop in cervical epithelial cells to maintain homeostasis of cervical tissue. Intriguingly, we found that high-risk HPV targets LATS2 to disrupt the feedback loop leading to the malignant transformation of cervical epithelial cells. Finally, we report that mitomycin C, an FDA-approved drug that could upregulate LATS2 and drive cellular senescence in vitro and in vivo, induced a regression of cervical cancer in a pre-clinial animal model. Thus, high-risk HPV targeting the YAP1-LATS2 feedback loop represents a new mechanism of cervical cancer development.

Hippo Signaling in the Ovary: Emerging Roles in Development, Fertility, and Disease.

Emerging studies indicate that the Hippo pathway, a highly conserved pathway that regulates organ size control, plays an important role in governing ovarian physiology, fertility, and pathology. Specific to the ovary, the spatiotemporal expression of the major components of the Hippo signaling cascade are observed throughout the reproductive lifespan. Observations from multiple species begin to elucidate the functional diversity and molecular mechanisms of Hippo signaling in the ovary in addition to the identification of interactions with other signaling pathways and responses to various external stimuli. Hippo pathway components play important roles in follicle growth and activation, as well as steroidogenesis, by regulating several key biological processes through mechanisms of cell proliferation, migration, differentiation, and cell fate determination. Given the importance of these processes, dysregulation of the Hippo pathway contributes to loss of follicular homeostasis and reproductive disorders such as polycystic ovary syndrome (PCOS), premature ovarian insufficiency, and ovarian cancers. This review highlights what is currently known about the Hippo pathway core components in ovarian physiology, including ovarian development, follicle development, and oocyte maturation, while identifying areas for future research to better understand Hippo signaling as a multifunctional pathway in reproductive health and biology.

Anti-Müllerian Hormone Inhibits FSH-Induced Cumulus Oocyte Complex In Vitro Maturation and Cumulus Expansion in Mice.

Anti-Müllerian hormone (AMH) is secreted by the ovaries of female animals and exerts its biological effects through the type II receptor (AMHR2). AMH regulates follicular growth by inhibiting the recruitment of primordial follicles and reducing the sensitivity of antral follicles to FSH. Despite the considerable research on the actions of AMH in granulosa cells, the effect of AMH on the in vitro maturation of oocytes remains largely unknown. In the current study, we showed that AMH is only expressed in cumulus cells, while AMHR2 is produced in both cumulus cells and oocytes. AMH had no significant effect on COCs nuclear maturation, whereas it inhibited the stimulatory effects of FSH on COCs maturation and cumulus expansion. Moreover, AMH treatment effectively inhibited the positive effect of FSH on the mRNA expressions of Hyaluronan synthase 2 (Has2), Pentraxin 3 (Ptx3), and TNF-alpha-induced protein 6 (Tnfaip 6) genes in COCs. In addition, AMH significantly decreased the FSH-stimulated progesterone production, but did not change estradiol levels. Taken together, our results suggest that AMH may inhibit the effects of FSH-induced COCs in vitro maturation and cumulus expansion. These findings increase our knowledge of the functional role of AMH in regulating folliculogenesis.